CN108165642A - Aedes albopictus and Aedes aegypti CYTB gene identification reagent boxes and its discrimination method based on high-resolution melting curve - Google Patents
Aedes albopictus and Aedes aegypti CYTB gene identification reagent boxes and its discrimination method based on high-resolution melting curve Download PDFInfo
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- CN108165642A CN108165642A CN201810273284.XA CN201810273284A CN108165642A CN 108165642 A CN108165642 A CN 108165642A CN 201810273284 A CN201810273284 A CN 201810273284A CN 108165642 A CN108165642 A CN 108165642A
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention relates to a kind of species identification fields, aedes albopictus and Aedes aegypti identification reagent box based on being a kind of melting curve by high-resolution, its main feature is that designing universal primer according to aedes albopictus and Aedes aegypti CYTB gene orders, differentiate aedes albopictus and Aedes aegypti various forms such as adult, ovum, larva and pupa etc. using the high-resolution melting curve reaction system of optimization.Aedes albopictus and Aedes aegypti can be differentiated, easy to operate, speed is fast, PCR product without post processing, the difference that can detect single base, really realize stopped pipe operation.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to the lineae ablicantes based on a kind of melting curve by high-resolution
Yellow-fever mosquito and Aedes aegypti identification reagent box.
Background technology
Port Mosquito population identifies main means as Morphological Identification, reflects by having Morphological Identification experience and mosquito class form
The Check and Examination of Port quarantine functionary of capability is identified according to medicine mosquito class taxa key.However, Morphological Identification faces very
More problems:The mosquito class professional accreditation talent is few, and the personnel education period is long;Experience level, professional standards, the mosquito class of appraiser are complete
Property can cause qualification cycle long with factors such as stages of development, and efficiency is low;Some complicated population such as sibling species groups and similar shape population
It is difficult to identify;For mosquito class ovum, larva and pupa, to obtain its secured identification feature, it is often necessary to which mosquito class is identified after manually cultivating;
It is very big test to port mosquito class appraisal if inspection mosquito class quantity in port is larger;Etc..Thus, port mosquito class mirror
It is fixed other than improving quarantine functionary's identification capacity and research and development identification auxiliary system, it is still necessary to development safely, fast and accurately, it is sensitive
Identification method, to realize mosquito class Rapid identification.
In recent years, with the development of molecular biology technology, Standard PCR, restriction fragment length polymorphism
(RFLP), randomly amplified polymorphic DNA(RAPD), single-strand conformation polymorphism(SSCP), DNA bar code technology equimolecular means
Through being applied to Mosquito population identification.However, these identification methods have shortcoming:Conventional detection freeze-draw method sequence, according to
Molecular probe more expensive Lai Yu or product sequencing;RFLP identifications mosquito kind not only takes, but also need great amount of samples;RAPD identifies mosquito
Kind is quick, but unstable result, and sometimes not reproducible;SSCP can only will be determined as the method for detection mosquito genoid mutation
The position of mutation and type need to further be sequenced, and deposition condition requirement is stringenter;The follow-up sequencing of DNA bar code technology
Work takes, consumption funds.In view of the deficiency of the above method, in order to safely, fast and accurately, sensitively realize point of Mosquito population
Son identification, needs to seek new Molecular Identification means.
Invention content
For the disadvantages described above or Improvement requirement of the prior art, the present invention provides one kind using high-resolution melting curve as base
The aedes albopictus and Aedes aegypti identification reagent box of plinth can be new as aedes albopictus and Aedes aegypti discriminating means, it is with monokaryon
Thuja acid melting temperature is different and is formed based on different shape melting curve and carries out mosquito class identification, it is easy to operate, speed is fast, it is low into
Originally, high-throughput, accurate, the not examined site of result limitation, PCR products are without post-processing, can detect single base
The advantages such as difference realize that real stopped pipe operation so as to reduce pollution risk, is very suitable for the analysis of a large amount of samples.
The present invention is realized by following technical scheme:
The present invention devises one group of aedes albopictus and Aedes aegypti diagnostic primers based on high-resolution melting curve, sequence
It is as follows:
CYTB-F:5’-GCTTGTTTATTTATTCACGTA-3’.
CYTB-R:5’-AATCCTCCTCAAATTCATTG-3’.
Using above-mentioned detection primer, the present invention a kind of melting curve by high-resolution is provided based on aedes albopictus and
Aedes aegypti identification reagent box, it includes such as ingredient:
1)The universal primer of aedes albopictus and Aedes aegypti ATP6 genes
CYTB-F:5’-GCTTGTTTATTTATTCACGTA-3’.
CYTB-R:5’-AATCCTCCTCAAATTCATTG-3’.
2)PCR mixed liquors:FastStart Taq archaeal dna polymerases, reaction buffer, dNTP mixed liquors, ResoLight satisfy
And fluorescent dye.
3)Aedes albopictus and Aedes aegypti genome.
4)ddH2O。
5)25 mM MgCl2。
Wherein, the reaction buffer is PCR buffer (10X) (PCR reaction buffers), is that this field is common
Commercialization buffer solution.
The present invention also provides the aedes albopictus and Aedes aegypti discrimination method based on a kind of melting curve by high-resolution,
It is characterized in that it includes the following steps:
2)The preparation of high-resolution melting curve template aedes albopictus and Aedes aegypti genomic DNA
It is organized using electric homogenizer cryogrinding aedes albopictus and Aedes aegypti.Use TaKaRa MiniBEST
Universal Genomic DNA Extraction Kit Ver.5.0 kits (TaKaRa companies), with reference to kit explanation
Extract aedes albopictus and Aedes aegypti genomic DNA.
3)High-resolution melting curve reaction system:
30 μ L reaction systems include:CYTB-F 0.6 μL(10 μm of working concentrations), 0.6 μ L of CYTB-R(10 μm of work are dense
Degree), 2 μ L of template, 3 μ L, FastStart Taq archaeal dna polymerases of reaction buffer, 0.5 μ L, dNTP mixed liquor, 2 μ L,
MgCl2 3.6 μ L, ResoLight saturated fluorescence dyestuff 1 μ L, ddH2O 16.7 μL。
4)High-resolution melting curve response procedures:
94 DEG C of 5 min of pre-degeneration.94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle.72 DEG C of extensions
5min。
High-resolution melting curve is directly run after PCR.95 DEG C of denaturation 1min.40 DEG C of cooling 1min.65-95℃
Continuous warming(25 fluorescence signals are collected in 1 DEG C of heating every time).40 DEG C of cooling 10s.Use 480 Gene of LightCycler
Scanning Software softwares analyze high-resolution melting curve.
The present invention a kind of melting curve by high-resolution based on aedes albopictus and Aedes aegypti identification reagent box and
Operating method designs universal primer according to aedes albopictus and Aedes aegypti CYTB gene orders, can to aedes albopictus and it is Egyptian she
Adult, ovum, larva and the pupa of mosquito are differentiated, compared with existing discriminating means, advantage of the invention is that:
1)The various states of aedes albopictus and Aedes aegypti can be differentiated, such as adult, ovum, larva and pupa state, be not required to
Differentiate after hatching.
2)The primer that the present invention designs, high sensitivity, high specificity.
3)It is easy to operate, speed is fast, the same day can provide result.
4)PCR products realize real stopped pipe behaviour without advantages such as post processing, the differences that can detect single base
Make so as to reduce pollution risk, be very suitable for the analysis of a large amount of samples.
Description of the drawings
Fig. 1:Aedes albopictus and Aedes aegypti CYTB PCR result figures, wherein, M:DL2000,1 aedes albopictus, 2 it is Egyptian she
Mosquito.
Fig. 2:Aedes aegypti CYTB genes relative to aedes albopictus CYTB genes high-resolution melting curve figure.(Horizontal seat
Temperature is designated as, ordinate is relative intensity of fluorescence), wherein the curve for having fluctuation is Aedes aegypti, straight line is aedes albopictus.
Specific embodiment
Below with embodiment, the invention will be further described.
The present embodiment provides the aedes albopictus and Aedes aegypti identification reagent based on a kind of melting curve by high-resolution
Box, including following ingredient:
1)The universal primer of aedes albopictus and Aedes aegypti CYTB genes
CYTB-F:5’-GCTTGTTTATTTATTCACGTA-3’
CYTB-R:5’-AATCCTCCTCAAATTCATTG-3’
2)PCR mixed liquors:FastStart Taq archaeal dna polymerases, reaction buffer, dNTP mixed liquors, ResoLight saturations are glimmering
Photoinitiator dye
3)Aedes albopictus and Aedes aegypti genome
4)ddH2O
5)25 mM MgCl2
Aedes albopictus and Aedes aegypti discrimination method based on additionally providing a kind of melting curve by high-resolution, by following
Step carries out:
1)It is organized using electric homogenizer cryogrinding aedes albopictus and Aedes aegypti.Use TaKaRa MiniBEST
Universal Genomic DNA Extraction Kit Ver.5.0 kits (TaKaRa companies), with reference to kit explanation
Extract aedes albopictus and Aedes aegypti genomic DNA.
2)30 μ L high-resolution melting curve reaction systems include:CYTB-F 0.6 μL(10 μm of working concentrations), CYTB-
R 0.6 μL(10 μm of working concentrations), 2 μ L of template, 3 μ L, FastStart Taq archaeal dna polymerases of reaction buffer, 0.5 μ
L, dNTP mixed liquor 2 μ L, MgCl2 3.6 μ L, ResoLight saturated fluorescence dyestuff 1 μ L, ddH2O 16.7 μL。
3)High-resolution melting curve response procedures:94 DEG C of 5 min of pre-degeneration.94 DEG C of denaturation 30s, 52 DEG C of annealing 30s,
72 DEG C of extension 1min, totally 30 recycle.72 DEG C of extension 5min.High-resolution melting curve is directly run after PCR.95 DEG C of changes
Property 1min.40 DEG C of cooling 1min.65-95 DEG C of continuous warming(25 fluorescence signals are collected in 1 DEG C of heating every time).40 DEG C of coolings
10s.High-resolution melting curve is analyzed using 480 Gene Scanning Software softwares of LightCycler.
4)As a result:Such as Fig. 1 and Fig. 2, aedes albopictus and Aedes aegypti CYTB PCR amplify band, and amplified fragments are long
230bp is spent, gene sequencing is simultaneously compared with NCBI Blast, and comparison result is corresponding aedes albopictus and Aedes aegypti.It utilizes
480 Gene Scanning Software softwares of LightCycler analyze high-resolution melting curve, find lineae ablicantes she
The curve difference of mosquito and Aedes aegypti is apparent, can effectively distinguish.
5)Conclusion:The present invention differentiates aedes albopictus and Aedes aegypti using high-resolution fusion curve for the first time, has expanded mosquito class
The visual angle of identification.The present invention is also that mosquito kind is reflected using the high-resolution fusion curve based on aedes albopictus and Aedes aegypti CYTB genes
A fixed new direction.This kit is easy to operate, speed is fast, PCR products are without post-processing, can detect single base
Difference, really realize stopped pipe operation, the various states of aedes albopictus and Aedes aegypti can be differentiated, such as adult, ovum, children
The states such as worm and pupa differentiate after not needing to hatching.
Certainly, it is limitation of the present invention that above description, which is not, and the present invention is also not limited to the example above, the art
Those of ordinary skill, in the essential scope of the present invention, the variations, modifications, additions or substitutions made should all belong to the present invention
Protection domain.
Sequence table
<110>Shandong international travel health care center
<120>Aedes albopictus and Aedes aegypti CYTB gene identification reagent boxes based on high-resolution melting curve and its
Discrimination method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gcttgtttat ttattcacgt a 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aatcctcctc aaattcattg 20
Claims (3)
1. aedes albopictus and Aedes aegypti diagnostic primers based on high-resolution melting curve, it is characterized in that, sequence is as follows:
CYTB-F:5’-GCTTGTTTATTTATTCACGTA-3’
CYTB-R:5’-AATCCTCCTCAAATTCATTG-3’.
2. aedes albopictus and Aedes aegypti identification reagent box based on high-resolution melting curve, it is characterized in that, it includes
Following ingredient:
1)The universal primer of aedes albopictus and Aedes aegypti CYTB genes
CYTB-F:5’-GCTTGTTTATTTATTCACGTA-3’;
CYTB-R:5’-AATCCTCCTCAAATTCATTG-3’;
2)PCR mixed liquors:FastStart Taq archaeal dna polymerases, reaction buffer, dNTP mixed liquors, ResoLight saturations are glimmering
Photoinitiator dye;
3)Aedes albopictus and Aedes aegypti genome;
4)ddH2O;
5)25 mM MgCl2。
3. aedes albopictus and Aedes aegypti discrimination method based on a kind of melting curve by high-resolution, it is characterized in that, it is wrapped
Include following steps:
1)The preparation of high-resolution melting curve template aedes albopictus and Aedes aegypti genomic DNA
It is organized using electric homogenizer cryogrinding aedes albopictus and Aedes aegypti;Use TaKaRa MiniBEST
Universal Genomic DNA Extraction Kit Ver.5.0 kits (TaKaRa companies), with reference to kit explanation
Extract aedes albopictus and Aedes aegypti genomic DNA;
2)High-resolution melting curve reaction system:
30 μ L reaction systems include:CYTB-F 0.6 μL(10 μm of working concentrations), 0.6 μ L of CYTB-R(10 μm of work are dense
Degree), 2 μ L of template, 3 μ L, FastStart Taq archaeal dna polymerases of reaction buffer, 0.5 μ L, dNTP mixed liquor, 2 μ L,
MgCl2 3.6 μ L, ResoLight saturated fluorescence dyestuff 1 μ L, ddH2O 16.7 μL;
3)High-resolution melting curve response procedures:
94 DEG C of 5 min of pre-degeneration;94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle;72 DEG C of extensions
5min;
High-resolution melting curve is directly run after PCR;95 DEG C of denaturation 1min;40 DEG C of cooling 1min;65-95 DEG C of continuous liter
Temperature(25 fluorescence signals are collected in 1 DEG C of heating every time);40 DEG C of cooling 10s;Use 480 Gene of LightCycler
Scanning Software softwares analyze high-resolution melting curve.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109321566A (en) * | 2018-08-20 | 2019-02-12 | 烟台国际旅行卫生保健中心 | A method of it expanding the specific primer of aedes albopictus Cytb gene and is sequenced using it |
CN109735535A (en) * | 2018-08-20 | 2019-05-10 | 烟台国际旅行卫生保健中心 | A method of it expanding Dulicidae insect Cytb gene-specific primer and is sequenced using it |
CN110885890A (en) * | 2019-11-29 | 2020-03-17 | 广西壮族自治区农业科学院植物保护研究所 | HRM primer, kit and method for rapidly identifying Bactrocera cucurbitae and Bactrocera south Asia |
CN111334583A (en) * | 2020-01-19 | 2020-06-26 | 中国检验检疫科学研究院 | Method for identifying aedes species based on PCR-MS mass spectrometry scanning |
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CN105039563A (en) * | 2015-08-14 | 2015-11-11 | 珠海国际旅行卫生保健中心 | Probe identifying three aedes simultaneously on the basis of gene chip and kit |
CN105039364A (en) * | 2015-08-14 | 2015-11-11 | 珠海国际旅行卫生保健中心 | Sequence of standard gene of DNA (deoxyribonucleic acid) barcode of aedes and application thereof |
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CN105039563A (en) * | 2015-08-14 | 2015-11-11 | 珠海国际旅行卫生保健中心 | Probe identifying three aedes simultaneously on the basis of gene chip and kit |
CN105039364A (en) * | 2015-08-14 | 2015-11-11 | 珠海国际旅行卫生保健中心 | Sequence of standard gene of DNA (deoxyribonucleic acid) barcode of aedes and application thereof |
Non-Patent Citations (1)
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109321566A (en) * | 2018-08-20 | 2019-02-12 | 烟台国际旅行卫生保健中心 | A method of it expanding the specific primer of aedes albopictus Cytb gene and is sequenced using it |
CN109735535A (en) * | 2018-08-20 | 2019-05-10 | 烟台国际旅行卫生保健中心 | A method of it expanding Dulicidae insect Cytb gene-specific primer and is sequenced using it |
CN110885890A (en) * | 2019-11-29 | 2020-03-17 | 广西壮族自治区农业科学院植物保护研究所 | HRM primer, kit and method for rapidly identifying Bactrocera cucurbitae and Bactrocera south Asia |
CN110885890B (en) * | 2019-11-29 | 2023-11-21 | 广西壮族自治区农业科学院植物保护研究所 | HRM primer, kit and method for rapidly identifying melon fly and Nanya fruit fly |
CN111334583A (en) * | 2020-01-19 | 2020-06-26 | 中国检验检疫科学研究院 | Method for identifying aedes species based on PCR-MS mass spectrometry scanning |
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